狂犬病病毒糖蛋白真核表达载体的构建及其在COS-7细胞中的表达

目的构建狂犬病病毒糖蛋白基因的真核细胞表达载体,并在COS-7细胞中表达。方法培养狂犬病病毒,提取负链RNA,采用RT-PCR的方法扩增糖蛋白基因,并将该基因克隆到真核表达载体pcDNA3.1(A)上,通过脂质体介导的方法,转染COS-7细胞,用ELISA及间接免疫荧光法检测狂犬病病毒糖蛋白在COS-7细胞中的瞬时表达。结果成功构建了真核表达载体pcDNA3.1(A)/G,转染COS-7细胞后,在其培养上清中未检测到狂犬病病毒糖蛋白的表达。间接免疫荧光试验表明,pcDNA3.1(A)/G能有效地表达狂犬病病毒糖蛋白于转染的COS-7细胞膜上。结论真核细胞表达载体pcDNA3.1(A)/G能够在COS-7细胞中表达狂犬病病毒糖蛋白,为开发狂犬病病毒糖蛋白基因工程疫苗奠定了基础。

Construction of Recombinant Plasmid for Expression of Rabies Virus Glycoprotein in COS-7 Cells

Objective To construct a recombinant plasmid for expression of rabies virus glycoprotein in COS-7 cells.Methods Culture rabies virus and extract negative strand RNA for amplification of glycoprotein gene by RT-PCR.Insert the amplified gene into eukaryotic expression vector pcDNA3.1(A) and transfect COS-7 cells with the constructed recombinant plasmid pcDNA3.1(A)/G in mediation of liposome.Determine the transient expression of rabies virus glycoprotein by ELISA and IFA.Results Restriction analysis showed successful construction of eukaryotic expression vector pcDNA3.1(A)/G.No expression of rabies virus glycoprotein was detected in the culture supernatant of transfected COS-7 cells.IFA proved the expression of rabies virus glycoprotein on surface of transfected COS-7 cells.Conclusion The constructed eukaryotic expression vector pcDNA3.1(A)/G was suitable for the expression of rabies virus glycoprotein in COS-7 cells,which laid a foundation of developing recombinant rabies virus glycoprotein vaccine.